Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Most research has focused on long-term tolerance, which occurs 24-72 hours following preconditioning and is mediated by changes in gene expression. In contrast we have focused on short-term ischemic tolerance, which is mediated by rapid biochemical events and occurs 1 hour following the preconditioning. The identification of the mechanisms of short-term tolerance may identify novel rapid acting therapeutic targets to treat acute ischemic episodes (stroke). Our studies have suggested the ubiquitin-proteasome system, which regulates protein degradation, as being responsible for rapid ischemic tolerance. Using a proteomics screen we identified 2 candidate proteins, fascin and myristilated alanine-rich C kinase substrate (MARCKS), which are selectively ubiquitinated and degraded following preconditioning ischemia. The degradation of these candidate proteins results in the reorganization of the actin cytoskeleton, and may reduce NMDA receptor function, which is implicated in excitotoxic cell injury following ischemia. Our central hypothesis is the degradation of fascin and MARCKS following preconditioning ischemia, results in brief cytoskeleton re-arrangement and disruption of NMDA receptor function thereby protecting the brain from harmful ischemia. Our hypothesis will be tested using an in vitro model of ischemia. In Aim 1, Investigate the effect of preconditioning ischemia on cytoskeletal protein degradation by the ubiquitin-proteasome system, we will investigate the role of the ubiquitin-proteasome system on the rapid degradation of our two candidate proteins, fascin and MARCKS, following preconditioning ischemia. In the Aim 2, Investigate changes in postsynaptic structural reorganization in ischemic tolerance, we will study the effect of preconditioning ischemia on the reorganization of the postsynaptic membrane. Experiments will address the effect of preconditioning ischemia on the structure of dendrites. We will determine the relevance of actin reorganization on ischemic tolerance by using actin stabilizing/ destabilizing compounds. Using co- immunoprecipitation we will investigate actin-NMDA receptor interactions following preconditioning. Understanding how preconditioning remodels synaptic structure and function may help identify harmful vs. protective mechanisms induced by brief ischemia in the brain, and give a clearer understanding of brain cell death. We believe that the rapid and selective degradation of specific brain proteins induces a protective state and may reveal suitable targets for pharmacological therapeutics. Indeed, the long-term aim of these studies is to discover endogenous protective mechanisms that can be translated into effective rapid acting neuroprotective agents for stroke. [unreadable] [unreadable] [unreadable]